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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55870完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲(Jing-Jer Lin) | |
| dc.contributor.author | Shao-Jung Yen | en |
| dc.contributor.author | 顏劭容 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:09:51Z | - |
| dc.date.available | 2014-10-09 | |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55870 | - |
| dc.description.abstract | 四股螺旋結構 (G-quadruplexs) 是由含有許多重複鳥糞嘌呤 (Guanine) 的DNA序列所形成， G-quadruplexs二級結構容易穩定的在端粒和一些啟動子區域的原致癌基因上形成，我們發現WNT1啟動子上帶有一段G-rich的序列，利用可穩定G-quadruplex結構的化合物BMVC和BMVC4，這兩種G-quadruplex stabilizers藥物可以幫助WNT1啟動子上的G-quadruplex形成，並抑制下游WNT1基因的表現，為了進一步了解是否抑制的現象是經由G-quadruplex的結構，我們利用點突變的序列做in vitro (CD and NMR)的分析以及螢光reporter的分析，來看wild-type和mutants的WNT1啟動子活性，從實驗結果中，的確G-quadruplex stabilizers可以抑制wild-type WNT1 promoter的活性，且顯示是屬於dose-dependent的方式，而mutants則不受藥物影響是因為不會形成G-quadruplex結構，除此之外，為了了解是否藥物影響Wnt1蛋白的表現是因改變蛋白質的結合，在ChIP分析中，Nucleolin結合到G-quadruplex的量有增加，顯示WNT1啟動子在藥物的處理下會形成穩定的G-quadruplex結構，而抑制Wnt1蛋白的表現。另外我們想知道是否藥物可以可以影響到其他啟動子G-quadruplex的形成，例如BMP2和EGFR啟動子，從微陣列基因分析BMP2的表現會被抑制，因此我們想知道透過G-quadruplex stabilizers抑制Bmp2是否可使EMT過程轉變，實驗指出Bmp2及EMT現象不受到G-quadruplex stabilizers影響，由於藥物無法作用到BMP2啟動子而使G-quadruplex結構無法形成；EGFR啟動子帶有兩段GFS，亦有機會形成G-quadruplex，因此我們利用螢光reporter分析GFS的功能為何，以分析是否藥物可以影響到EGFR的表現，在NMR氫圖譜證明-137 bps和-376 bps的GFS在in vitro下有形成G-quadruplex的訊號，從repoter分析結果中，藥物處理下可增加-137 bps GFS的EGFR啟動子之活性。 | zh_TW |
| dc.description.abstract | DNA sequences with tandem guanine repeats tend to from G-quadruplex (G4) structure. G-quadruplex is easily formed in telomere and the promoter regions of many proto-oncogenes. Previously our lab has found a G-rich sequences located at the WNT1 promoter is capable of forming G-quadruplex. The expression of Wnt1 was repressed by G-quadruplex stabilizers. To determine whether the repression is G-quadruplex structure dependent, we first analyzed mutations on these G-rich sequences using CD and NMR. Mutants that failed to form G-quadruplex were identified. I then established a luciferase reporter assay to monitor the WNT1 promoter expression. Our results revealed that wild-type WNT1 promoter can be suppressed by G-quadruplex stabilizers in a dose-dependent manner. Mutants that failed to form G-quadruplex structure were not repressed by drugs. The binding of nucleolin is also tested to check whether the repression of expression by BMVC is due to a change in protein binding. Using ChIP assay, we found BMVC increased the binding of nucleolin to WNT1 promoter, suggesting that the G-quadruplex structure was formed at the WNT1 promoter upon drug treatment. Thus, our results indicated that formation of the G-quadruplex structure at WNT1 promoter repressed its expression. We also identified G-qaudruplex-forming sequences on BMP2 and EGFR promoters. From microarray results, we found BMP2 is repressed by G-quadruplex stabilizers. Our results showed Bmp2 is not repressed due to G-quadruplex structure cannot be formed in cell level, suggesting our drugs cannot target to BMP2 promoter. We determine two G-qaudruplex-forming sequences on EGFR promoter functional roles by luciferase reporter assay and the ability to form G-quadruplex in vitro. And it can induce -137 GFS EGFR promoter activity in G-quadruplex stabilizers treament. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:09:51Z (GMT). No. of bitstreams: 1 ntu-103-R01442017-1.pdf: 3039299 bytes, checksum: e85081a06685deda7b2399814983174a (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 口試委員會審定書．．．．．．．．．．．．．．．．．1
致謝詞．．．．．．．．．．．．．．．．．．．．2 中文摘要．．．．．．．．．．．．．．．．．．．．4 英文摘要．．．．．．．．．．．．．．．．．．．．．．．5 目錄．．．．．．．．．．．．．．．．．．．．．．．．．．6 附圖目錄．．．．．．．．．．．．．．．．．．．．．．．7 縮寫表．．．．．．．．．．．．．．．．．．．．．．．．9 一、緒論．．．．．．．．．．．．．．．．．．．．．．11 二、實驗材料與方法．．．．．．．．．．．．．．．．．．18 三、實驗結果．．．．．．．．．．．．．．．．．．．．．．．27 四、討論．．．．．．．．．．．．．．．．．．．．．．．．35 五、參考文獻．．．．．．．．．．．．．．．．．．．．．．38 六、附圖．．．．．．．．．．．．．．．．．．．．．．．42 | |
| dc.language.iso | zh-TW | |
| dc.title | "分析WNT-1, BMP2及EGFR啟動子之G-quadruplex形成序列" | zh_TW |
| dc.title | Analyzing the role of G-quadruplex-forming sequence on WNT-1, BMP2 and EGFR promoter | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張大釗(Ta-Chau Chang),婁培人(Pei-Jen Lou) | |
| dc.subject.keyword | 四股螺旋結構, | zh_TW |
| dc.subject.keyword | G-quadruplex,BMVC,BMVC4, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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